CN113912676B - 一种酒糟黄鱼抗菌肽fah34及其应用 - Google Patents
一种酒糟黄鱼抗菌肽fah34及其应用 Download PDFInfo
- Publication number
- CN113912676B CN113912676B CN202111261773.1A CN202111261773A CN113912676B CN 113912676 B CN113912676 B CN 113912676B CN 202111261773 A CN202111261773 A CN 202111261773A CN 113912676 B CN113912676 B CN 113912676B
- Authority
- CN
- China
- Prior art keywords
- fah34
- antibacterial peptide
- antibacterial
- peptide
- yellow croaker
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
- 239000003910 polypeptide antibiotic agent Substances 0.000 title claims abstract description 74
- 241001596950 Larimichthys crocea Species 0.000 title claims abstract description 26
- 241000607272 Vibrio parahaemolyticus Species 0.000 claims abstract description 31
- 230000002401 inhibitory effect Effects 0.000 claims abstract description 21
- 230000000844 anti-bacterial effect Effects 0.000 claims abstract description 13
- 125000003275 alpha amino acid group Chemical group 0.000 claims abstract description 12
- 239000003674 animal food additive Substances 0.000 claims abstract description 8
- 239000005452 food preservative Substances 0.000 claims abstract description 8
- 235000019249 food preservative Nutrition 0.000 claims abstract description 8
- 239000003814 drug Substances 0.000 claims abstract 2
- 230000002147 killing effect Effects 0.000 claims description 10
- 239000003242 anti bacterial agent Substances 0.000 claims description 5
- 229940124350 antibacterial drug Drugs 0.000 claims description 4
- 239000004480 active ingredient Substances 0.000 claims 3
- 230000000694 effects Effects 0.000 abstract description 9
- 238000011282 treatment Methods 0.000 abstract description 6
- 208000010824 fish disease Diseases 0.000 abstract description 2
- 230000012010 growth Effects 0.000 abstract description 2
- 208000015181 infectious disease Diseases 0.000 abstract description 2
- 108090000765 processed proteins & peptides Proteins 0.000 description 18
- 102000044503 Antimicrobial Peptides Human genes 0.000 description 17
- 108700042778 Antimicrobial Peptides Proteins 0.000 description 17
- 241000894006 Bacteria Species 0.000 description 14
- 210000000170 cell membrane Anatomy 0.000 description 9
- 102000004196 processed proteins & peptides Human genes 0.000 description 9
- 230000001580 bacterial effect Effects 0.000 description 7
- 239000000126 substance Substances 0.000 description 7
- 238000010438 heat treatment Methods 0.000 description 6
- 239000000047 product Substances 0.000 description 6
- 244000052616 bacterial pathogen Species 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 229920001184 polypeptide Polymers 0.000 description 5
- 208000019331 Foodborne disease Diseases 0.000 description 4
- 241000282414 Homo sapiens Species 0.000 description 4
- 230000006378 damage Effects 0.000 description 4
- 235000013305 food Nutrition 0.000 description 4
- 230000036541 health Effects 0.000 description 4
- 238000000034 method Methods 0.000 description 4
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 206010016952 Food poisoning Diseases 0.000 description 3
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 3
- 102000007079 Peptide Fragments Human genes 0.000 description 3
- 108010033276 Peptide Fragments Proteins 0.000 description 3
- 230000009471 action Effects 0.000 description 3
- 229940088710 antibiotic agent Drugs 0.000 description 3
- 230000003385 bacteriostatic effect Effects 0.000 description 3
- 230000005540 biological transmission Effects 0.000 description 3
- 238000002474 experimental method Methods 0.000 description 3
- 230000005764 inhibitory process Effects 0.000 description 3
- 244000144972 livestock Species 0.000 description 3
- 238000005259 measurement Methods 0.000 description 3
- 235000013372 meat Nutrition 0.000 description 3
- 244000000010 microbial pathogen Species 0.000 description 3
- 108090000623 proteins and genes Proteins 0.000 description 3
- 102000004169 proteins and genes Human genes 0.000 description 3
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 2
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 2
- 241000607142 Salmonella Species 0.000 description 2
- 150000001413 amino acids Chemical class 0.000 description 2
- 230000003698 anagen phase Effects 0.000 description 2
- 238000003556 assay Methods 0.000 description 2
- 238000012258 culturing Methods 0.000 description 2
- 235000013365 dairy product Nutrition 0.000 description 2
- 238000007865 diluting Methods 0.000 description 2
- 230000003834 intracellular effect Effects 0.000 description 2
- 238000004949 mass spectrometry Methods 0.000 description 2
- 238000001819 mass spectrum Methods 0.000 description 2
- 235000013622 meat product Nutrition 0.000 description 2
- 239000012528 membrane Substances 0.000 description 2
- 230000004048 modification Effects 0.000 description 2
- 238000012986 modification Methods 0.000 description 2
- 230000001717 pathogenic effect Effects 0.000 description 2
- 239000008363 phosphate buffer Substances 0.000 description 2
- 239000008055 phosphate buffer solution Substances 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 238000012216 screening Methods 0.000 description 2
- 241000251468 Actinopterygii Species 0.000 description 1
- 101100379209 Arabidopsis thaliana APD3 gene Proteins 0.000 description 1
- 241000193755 Bacillus cereus Species 0.000 description 1
- 241000589876 Campylobacter Species 0.000 description 1
- 241000193155 Clostridium botulinum Species 0.000 description 1
- 208000003322 Coinfection Diseases 0.000 description 1
- RYGMFSIKBFXOCR-UHFFFAOYSA-N Copper Chemical compound [Cu] RYGMFSIKBFXOCR-UHFFFAOYSA-N 0.000 description 1
- 206010059866 Drug resistance Diseases 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- 208000005577 Gastroenteritis Diseases 0.000 description 1
- 241000186779 Listeria monocytogenes Species 0.000 description 1
- DLCAXBGXGOVUCD-PPCPHDFISA-N Lys-Thr-Ile Chemical compound [H]N[C@@H](CCCCN)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O DLCAXBGXGOVUCD-PPCPHDFISA-N 0.000 description 1
- 241001465754 Metazoa Species 0.000 description 1
- KZPRPBLHYMZIMH-MXAVVETBSA-N Ser-Phe-Ile Chemical compound [H]N[C@@H](CO)C(=O)N[C@@H](CC1=CC=CC=C1)C(=O)N[C@@H]([C@@H](C)CC)C(O)=O KZPRPBLHYMZIMH-MXAVVETBSA-N 0.000 description 1
- 241000191967 Staphylococcus aureus Species 0.000 description 1
- 238000003917 TEM image Methods 0.000 description 1
- ABUBSBSOTTXVPV-UHFFFAOYSA-H [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O Chemical compound [U+6].CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O.CC([O-])=O ABUBSBSOTTXVPV-UHFFFAOYSA-H 0.000 description 1
- 239000002253 acid Substances 0.000 description 1
- 238000004458 analytical method Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- HOQPTLCRWVZIQZ-UHFFFAOYSA-H bis[[2-(5-hydroxy-4,7-dioxo-1,3,2$l^{2}-dioxaplumbepan-5-yl)acetyl]oxy]lead Chemical compound [Pb+2].[Pb+2].[Pb+2].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O.[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O HOQPTLCRWVZIQZ-UHFFFAOYSA-H 0.000 description 1
- 238000009835 boiling Methods 0.000 description 1
- 238000009395 breeding Methods 0.000 description 1
- 230000001488 breeding effect Effects 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 230000008859 change Effects 0.000 description 1
- 239000003153 chemical reaction reagent Substances 0.000 description 1
- 238000011109 contamination Methods 0.000 description 1
- 229910052802 copper Inorganic materials 0.000 description 1
- 239000010949 copper Substances 0.000 description 1
- 230000001086 cytosolic effect Effects 0.000 description 1
- 230000018044 dehydration Effects 0.000 description 1
- 238000006297 dehydration reaction Methods 0.000 description 1
- 230000001066 destructive effect Effects 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 201000010099 disease Diseases 0.000 description 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 1
- 235000013601 eggs Nutrition 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 230000000415 inactivating effect Effects 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 230000001788 irregular Effects 0.000 description 1
- 235000021190 leftovers Nutrition 0.000 description 1
- 108010034529 leucyl-lysine Proteins 0.000 description 1
- 230000000670 limiting effect Effects 0.000 description 1
- 238000004811 liquid chromatography Methods 0.000 description 1
- 238000004519 manufacturing process Methods 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- 230000007246 mechanism Effects 0.000 description 1
- 230000000813 microbial effect Effects 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 230000008520 organization Effects 0.000 description 1
- 230000035699 permeability Effects 0.000 description 1
- 239000012071 phase Substances 0.000 description 1
- 235000013594 poultry meat Nutrition 0.000 description 1
- 230000002265 prevention Effects 0.000 description 1
- 230000008569 process Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000002829 reductive effect Effects 0.000 description 1
- 238000010532 solid phase synthesis reaction Methods 0.000 description 1
- 239000000243 solution Substances 0.000 description 1
- 239000006228 supernatant Substances 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000004627 transmission electron microscopy Methods 0.000 description 1
- 229910021642 ultra pure water Inorganic materials 0.000 description 1
- 239000012498 ultrapure water Substances 0.000 description 1
- 238000012795 verification Methods 0.000 description 1
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 1
- 238000005303 weighing Methods 0.000 description 1
- 235000020985 whole grains Nutrition 0.000 description 1
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
- C07K7/04—Linear peptides containing only normal peptide links
- C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K20/00—Accessory food factors for animal feeding-stuffs
- A23K20/10—Organic substances
- A23K20/142—Amino acids; Derivatives thereof
- A23K20/147—Polymeric derivatives, e.g. peptides or proteins
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23K—FODDER
- A23K50/00—Feeding-stuffs specially adapted for particular animals
- A23K50/80—Feeding-stuffs specially adapted for particular animals for aquatic animals, e.g. fish, crustaceans or molluscs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L3/00—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs
- A23L3/34—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals
- A23L3/3454—Preservation of foods or foodstuffs, in general, e.g. pasteurising, sterilising, specially adapted for foods or foodstuffs by treatment with chemicals in the form of liquids or solids
- A23L3/3463—Organic compounds; Microorganisms; Enzymes
- A23L3/3526—Organic compounds containing nitrogen
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Polymers & Plastics (AREA)
- Health & Medical Sciences (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Organic Chemistry (AREA)
- Medicinal Chemistry (AREA)
- Zoology (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Animal Husbandry (AREA)
- General Chemical & Material Sciences (AREA)
- General Health & Medical Sciences (AREA)
- Oncology (AREA)
- Public Health (AREA)
- Microbiology (AREA)
- Genetics & Genomics (AREA)
- Communicable Diseases (AREA)
- Biophysics (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Molecular Biology (AREA)
- Veterinary Medicine (AREA)
- Insects & Arthropods (AREA)
- Marine Sciences & Fisheries (AREA)
- Biochemistry (AREA)
- Birds (AREA)
- Nutrition Science (AREA)
- Peptides Or Proteins (AREA)
- Food Preservation Except Freezing, Refrigeration, And Drying (AREA)
- Agricultural Chemicals And Associated Chemicals (AREA)
Abstract
本发明公开了一种酒糟黄鱼抗菌肽FAH34,其氨基酸序列为KTISFILK,抗菌肽的分子量为949道尔顿。本发明抗菌肽具有强烈的抑制副溶血性弧菌生长的作用,且在经过高温处理后依旧具有良好的抑菌活性,可作为制备预防或抑制副溶血性弧菌感染的药物。本发明为后续进一步研究酒糟黄鱼抗菌肽作为食品防腐剂和防止鱼病的饲料添加剂开发奠定了基础。
Description
技术领域
本发明涉及生物技术领域,尤其涉及一种酒糟黄鱼抗菌肽FAH34及其应用。
背景技术
食源致病微生物是食品、食用农产品产供过程的关键潜在的生物危害因素,严重威胁食品、食用农产品质量安全,给人类和动物健康造成严重危害。据世界卫生组织(WHO)统计,全球每年约有15亿人感染食源性疾病,其中70%为致病性微生物污染所造成的。其中常见的致病微生物有:沙门氏菌(禽畜肉、蛋类)、弯曲菌(禽肉)、金黄色葡萄球菌(畜禽肉、乳品)、副溶血性弧菌(水产品)、蜡样芽孢杆菌(剩饭)、肉毒梭菌(发酵制品、肉制品)、单核细胞增生李斯特菌(乳制品、即食食品)以及致病性大肠杆菌(畜禽肉)等。而沙门氏菌以及副溶血性弧菌是沿海地区两种最常见的食源性致病菌,也是引起沿海地区人民食物中毒的首要食源性致病菌。
副溶血性弧菌广泛存在于海洋环境中,是引起食物中毒爆发的主要病原菌之一,也是一种人和海洋生物共感染的病原菌。既可引起人食物中毒、肠胃炎等,危害人体健康,又可引起水产品养殖病,造成经济损失。传统抗生素在致病微生物防控中发挥着极重要的作用,但在近年来,由于抗生素的滥用所带来的副作用及病菌抗药性问题也愈发凸显,严重影响了人类健康及生态安全。抗菌肽作为广泛存在于自然界生物中的一类多肽物质,对细菌具有良好的抑制作用,可作为传统抗生素和饲料添加剂的代替品。大部分抗菌肽具有热稳定性,在l00℃下加热10~15min仍能保持其活性,超过100℃仍能保持活性的抗菌肽则比较罕见。
因此,提供一种能有效抑制副溶血性弧菌感染,同时能保持良好的热稳定性的抗菌肽是本领域亟待解决的技术问题。
发明内容
本发明的目的在于提供一种酒糟黄鱼抗菌肽及其应用,通过酒糟黄鱼抗菌肽FAH34对副溶血性弧菌抑菌活性以及热稳定性研究,为寻找新的食品防腐剂和预防鱼病水产饲料添加剂奠定基础。
为了解决上述目的,本发明采用的技术方案如下:
本发明解决其技术问题所采用的技术方案之一是:提供了一种酒糟黄鱼蛋白抗菌肽FAH34,其氨基酸序列KTISFILK,如SEQ ID NO:1所示。所示抗菌肽FAH34的分子量为949道尔顿,所带电荷为+2,总疏水性比为50%。
抗菌肽FAH34是从如下原理对细菌造成破坏:一方面,抗菌肽肽FAH34具有的正电荷可与细菌细胞膜发生作用,增加细菌细胞膜成孔活性。另一方面,破坏细胞膜的同时改变细菌细胞膜的通透性,使细胞内物质外渗从而导致细菌死亡。
本发明解决其技术问题所采用的技术方案之二是:提供了一种酒糟黄鱼抗菌肽FAH34在制备抗菌药物中的应用,该抗菌药物用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之三是:提供了一种抗菌药物,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之四是:提供了一种水产饲料添加剂,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述水产饲料添加剂用于抑制和/或杀灭副溶血性弧菌。
本发明解决其技术问题所采用的技术方案之五是:提供了一种食品防腐剂,其有效成分包括酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQ ID NO:1。
进一步地,所述食品防腐剂用于抑制和/或杀灭副溶血性弧菌。
本发明的抗菌肽可以采用本领域技术人员已知的方法合成,例如固相合成,并采用本领域技术人员已知的方法进行纯化,例如高效液相色谱法。
实施本发明,具有如下有益效果:
本发明以酒糟黄鱼为研究对象,通过LCMS质谱结果对所的肽段进行生物信息学预测,发现一个全新氨基酸序列的多肽FAH34。研究多肽FAH34对副溶血性弧菌的抑菌活性;并以副溶血性弧菌为例利用透射电镜观察FAH34对其破坏程度;最后研究了121℃以及80℃两种高温处理后该多肽的抑菌活性,验证其耐热性。实验结果表明,该肽对副溶血性弧菌具有强烈的抑制作用,且在经过高温处理后依旧具有良好的抑菌活性。它的抑菌机理是首先吸附在细菌表面,然后破坏细菌的细胞膜,并抑制膜的生成使细胞内物质外渗,从而达到使细菌失活的作用。良好的热稳定性使其在食品防腐剂的应用方面具备一定的潜力。
附图说明
图1为抗菌肽FAH34的质谱图。
图2为抗菌肽FAH34的结构示意图。
图3为抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图4为抗菌肽FAH34时间杀伤曲线图。
(▲)空白对照;(■)MIC;(●)2MIC;
图5为副溶血性弧菌透射电镜图.
(A)空白对照组;(B)抗菌肽FAH34处理的副溶血性弧菌;
图6为80℃加热30min后抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图7为121℃加热15min后抗菌肽FAH34对副溶血性弧菌最低抑制浓度(MIC)测定对照图。其中,
A:抗菌肽浓度0μg/mL;
B:抗菌肽浓度3.90625μg/mL;
C:抗菌肽浓度7.8125μg/mL;
D:抗菌肽浓度15.625μg/mL;
E:抗菌肽浓度31.25μg/mL;
F:抗菌肽浓度62.5μg/mL。
图8为80℃加热30min以及121℃加热15min后抗菌肽FAH34时间杀伤曲线图。
具体实施方式
为了更好地理解本发明,下面结合实施例和附图对本发明做进一步的详细说明,但本领域技术人员了解,下述实施例不是对本发明保护范围的限制,任何在本发明基础上做出的改变和变化,都在本发明的保护范围之内。
下述实施例中所使用的实验方法如无特殊说明,均为常规方法。
下述实施例中所用的材料、试剂等,如无特殊说明,均可从商业途径得到。
实施例1:酒糟黄鱼的液相色谱/质谱联用技术(LCMS)
称取100~200g酒糟黄鱼样品鱼肉于烧杯中,加入适量超纯水用搅拌机破碎3~5min,然后将破碎的组织于100℃水浴锅煮2~3h。在4℃条件下用11000g离心力离心20min制备样品。再将离心好的上清液过0-3000目筛后于冰箱隔夜冷冻后进行冻干。再将冻干后的样品进行LCMS实验。
色谱条件:进样量:5.0μl
色谱柱:C18分析色谱柱,长度25cm,内径75μm.
流动相:A:0.1%甲醇水溶液
B:乙腈
结合搜库软件:MAXQUANTv1.6.5.0、数据库:uniprot大黄鱼蛋白库对所得肽段质谱图进行区分鉴定,得到85种肽段。抗菌肽FAH34质谱结果如图1所示。
实施例2:酒糟黄鱼中抗菌肽的筛选
通过LCMS结果所得85种氨基酸序列,利用抗菌肽预测在线服务器APD3、CAMP对酒糟黄鱼蛋白序列中可能存在的抗菌序列进行预测,并对可能具有抗菌序列的电荷、疏水性进行分析,最终筛选出氨基酸序列KTISFILK化学合成(由北京中科亚光生物科技有限公司合成),并进行抑菌活性验证。筛选结果如下表所示。
表1酒糟黄鱼LCMS结果潜在抗菌肽的预测
从表1可以看出,序列KTISFILK具有典型抗菌肽的特征,即氨基酸数量(<50)、总正电荷(+2到+9)和疏水氨基酸百分比(>30%)。合成符合特征的2个肽(KTISFILK、AALDLLVPMKKNSNTAGGK),分别对副溶血性弧菌进行抑菌实验,结果显示肽序列KTISFILK的抑菌作用最强。
利用在线服务器I-TASSER对肽序列KTISFILK的结构进行预测,同时利用软件PyMOL观察其二级结构。结果如图2所示。
实施例3:最低抑制浓度(MIC)测定
将副溶血性弧菌在37℃培养12h至对数生长期,在0.01MpH7.2磷酸盐缓冲液中稀释至106-7CFU/mL。将肽溶于磷酸盐缓冲中,37℃等体积与菌混合处理2h。最低抑菌浓度(MIC)是指在37℃孵育过夜后,从微量滴定板上看不到细菌生长的抗菌肽最低浓度。如图3所示,FAH34对副溶血性弧的最低抑菌浓度(MIC)为3.90625μg/mL。
实施例4:时间杀伤曲线TIMEKILL测定
将副溶血性弧菌在37℃培养12h至对数生长期,在0.01MpH7.2磷酸盐缓冲液中稀释至103-4CFU/mL。取MIC和2×MIC浓度肽于37℃等体积与菌混合后分别进行孵育,每隔30分钟取样涂平板,37℃培养过夜后记录菌落总数。由结果可知,抗菌肽对副溶血性弧菌均在2.5h开始有明显效果;随后继续呈下降趋势。在抗菌肽作用下,细菌数量减少的更快。表明抗菌肽FAH34对副溶血性弧菌随着作用时间增加有明显的抑制效果(图4)。
实施例5:透射电镜分析
以106-7CFU/mL的细菌在37℃下用2×MIC的FAH34处理2h,然后在2700g下离心10min,用磷酸盐缓冲液(pH7.2)洗涤两次。用1%的锇酸固定后,用95%乙醇脱水,然后丙酮处理20min。样品在70℃下烘烤24h,在铜网格上制备70-90nm的薄片,然后用柠檬酸铅和乙酸铀酯染色。用H-7650透射电子显微镜观察和捕获超微结构。
如图5A所示,对于未经处理的细菌,细菌细胞的胞内组织和结构完整性良好。然而,如图5B所示,经过肽FAH34处理后,可以看到细菌细胞膜结构开始随着细胞空泡化而模糊,细胞的形状变得不规则,细胞膜完全塌陷,细胞质内容物流出。透射电子显微镜结果表明,FAH34肽对副溶血性弧菌的细胞膜和内部结构具有破坏作用。
实施例6:高温处理后抗菌肽FAH34的抑菌活性测定
分别对肽FAH34在121℃加热15min以及80℃加热30min,处理完成后对其最低抑菌浓度(MIC)以及时间杀灭动力学曲线TIMEKILL进行测定。测定方法同例3、例4中的测定方法。结果表明,在经过121℃加热15min以及80℃加热30min两种高温热处理后,肽FAH34保持了其抑菌活性,最低抑制浓度(MIC)和时间杀伤曲线(TIMEKILL)均未发生明显变化。如图6、7和8所示,经过热处理后的抗菌肽FAH34的最低抑菌浓度(MIC)仍为3.90625μg/mL,时间杀伤曲线(TIMEKILL)表明其在2.5h后有明显的杀菌效果。说明抗菌肽FAH34具有较好的热稳定性,可被用作热加工中的食品防腐剂以及水产饲料添加剂,在食品工业中具有良好的潜在应用前景。
综上,本发明提供了一种全新的抗菌肽FAH34,抗菌肽FAH34对副溶血弧菌的最低抑菌浓度(MIC)为3.90625μg/mL,对副溶血弧菌具有强烈的抑制作用,且具有较好的热稳定性。本发明抗菌肽FAH34是通过吸附在细菌表面,破坏细菌的细胞膜,并抑制膜的生成使细胞内物质外渗的原理而达到使细菌失活的作用。
虽然以上描述了本发明的具体实施方式,但是熟悉本技术领域的技术人员应当理解,我们所描述的具体的实施例只是说明性的,而不是用于对本发明的范围的限定,熟悉本领域的技术人员在依照本发明的精神所作的等效的修饰以及变化,都应当涵盖在本发明的权利要求所保护的范围内。
序列表
<110> 集美大学
<120> 一种酒糟黄鱼抗菌肽FAH34及其应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 8
<212> PRT
<213> Pseudosciaena crocea
<400> 1
Lys Thr Ile Ser Phe Ile Leu Lys
1 5
Claims (5)
1.一种酒糟黄鱼抗菌肽FAH34,其氨基酸序列如SEQIDNO:1所示。
2.如权利要求1所述的酒糟黄鱼抗菌肽FAH34在制备抗菌药物中的应用,其特征在于:所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
3.一种抗菌药物,其特征在于:其有效成分为酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQIDNO:1,所述抗菌药物用于抑制和/或杀灭副溶血性弧菌。
4.一种水产饲料添加剂,其特征在于:其有效成分为酒糟黄鱼抗菌肽FAH34,所述抗菌肽FAH34的氨基酸序列为SEQIDNO:1,所述水产饲料添加剂用于抑制和/或杀灭副溶血性弧菌。
5.一种食品防腐剂,其特征在于:其有效成分为酒糟黄鱼抗菌肽 FAH34,所述抗菌肽FAH34的氨基酸序列为SEQIDNO:1,所述食品防腐剂用于抑制和/或杀灭副溶血性弧菌。
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111261773.1A CN113912676B (zh) | 2021-10-28 | 2021-10-28 | 一种酒糟黄鱼抗菌肽fah34及其应用 |
Applications Claiming Priority (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CN202111261773.1A CN113912676B (zh) | 2021-10-28 | 2021-10-28 | 一种酒糟黄鱼抗菌肽fah34及其应用 |
Publications (2)
Publication Number | Publication Date |
---|---|
CN113912676A CN113912676A (zh) | 2022-01-11 |
CN113912676B true CN113912676B (zh) | 2023-02-17 |
Family
ID=79243369
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
CN202111261773.1A Active CN113912676B (zh) | 2021-10-28 | 2021-10-28 | 一种酒糟黄鱼抗菌肽fah34及其应用 |
Country Status (1)
Country | Link |
---|---|
CN (1) | CN113912676B (zh) |
Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108892718A (zh) * | 2018-07-03 | 2018-11-27 | 华南师范大学 | 一种斜带石斑鱼抗菌肽及其应用 |
CN111333700A (zh) * | 2020-03-23 | 2020-06-26 | 集美大学 | 一种大黄鱼乳清酸性蛋白抗菌肽及其应用 |
CN111333716A (zh) * | 2020-03-23 | 2020-06-26 | 集美大学 | 一种大黄鱼血红蛋白抗菌肽及其应用 |
CN112898386A (zh) * | 2021-03-02 | 2021-06-04 | 集美大学 | 一种大黄鱼肌球蛋白重链抗菌肽lcmhc及其应用 |
CN113185596A (zh) * | 2021-04-01 | 2021-07-30 | 集美大学 | 一种黄鱼鲞肌球蛋白抗菌肽lcm13及其应用 |
-
2021
- 2021-10-28 CN CN202111261773.1A patent/CN113912676B/zh active Active
Patent Citations (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108892718A (zh) * | 2018-07-03 | 2018-11-27 | 华南师范大学 | 一种斜带石斑鱼抗菌肽及其应用 |
CN111333700A (zh) * | 2020-03-23 | 2020-06-26 | 集美大学 | 一种大黄鱼乳清酸性蛋白抗菌肽及其应用 |
CN111333716A (zh) * | 2020-03-23 | 2020-06-26 | 集美大学 | 一种大黄鱼血红蛋白抗菌肽及其应用 |
CN112898386A (zh) * | 2021-03-02 | 2021-06-04 | 集美大学 | 一种大黄鱼肌球蛋白重链抗菌肽lcmhc及其应用 |
CN113185596A (zh) * | 2021-04-01 | 2021-07-30 | 集美大学 | 一种黄鱼鲞肌球蛋白抗菌肽lcm13及其应用 |
Also Published As
Publication number | Publication date |
---|---|
CN113912676A (zh) | 2022-01-11 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US10487112B2 (en) | Pediococcus acidilactici derived antimicrobial hexapeptide and preparation method thereof | |
CN112898386B (zh) | 一种大黄鱼肌球蛋白重链抗菌肽lcmhc及其应用 | |
CN111333716B (zh) | 一种大黄鱼血红蛋白抗菌肽及其应用 | |
Bi et al. | The antifungal mechanism of konjac flying powder extract and its active compounds against wood decay fungi | |
CN112760253A (zh) | 一种植物乳杆菌、抗菌肽及其应用 | |
CN111838308B (zh) | 一种可食性果蔬纳米涂膜保鲜剂及其制备方法和应用 | |
CN113185596B (zh) | 一种黄鱼鲞肌球蛋白抗菌肽lcm13及其应用 | |
CN110577910A (zh) | 一种侧孢短芽孢杆菌、抗菌脂肽及其在农业与食品上的应用 | |
CN113912676B (zh) | 一种酒糟黄鱼抗菌肽fah34及其应用 | |
KR101528193B1 (ko) | 락토바실러스 플란타룸 yml007로부터 분리된 항진균 활성을 갖는 펩타이드 | |
CN110724175B (zh) | 一种利用超声匀浆从紫贻贝加工下脚料中提取抗菌肽的制备方法 | |
CN113662035A (zh) | 一种冷鲜牛肉生物防腐剂的配方、制备方法及其使用方法 | |
CN113861271B (zh) | 一种黄鱼鲞风味肽Tit5及其应用 | |
Wang et al. | Bacillomycin D lipopeptides from marine Bacillus megaterium as antimicrobial and preservative agents for large yellow croaker, Larimichthys crocea | |
CN117986327B (zh) | 一种抗菌肽rs12及其应用 | |
CN107261113B (zh) | 天然宿主防御肽Alligatorin5的应用 | |
CN113024663B (zh) | 一种泥蚶血红蛋白α螺旋抗菌肽及其应用 | |
CN114409754B (zh) | 一种微电压方法协同抗菌肽pv-q5及其用途 | |
CN101849608B (zh) | 油菜饼抗菌肽 | |
CN114380893B (zh) | 一种腌制凡纳滨对虾抗菌肽ec-v10及其用途 | |
CN118047841B (zh) | 一种发酵桑叶抗菌肽Squ8及其应用 | |
Kitsiou et al. | On the evaluation of the antimicrobial effect of grape seed extract and cold atmospheric plasma on the dynamics of Listeria monocytogenes in novel multiphase 3D viscoelastic models | |
CN115710305A (zh) | 一种发酵大黄鱼琥珀酸脱氢蛋白抗菌肽sdh73及其应用 | |
CN112674199B (zh) | 一种多肽食品防腐剂及其制备方法 | |
CN114315972B (zh) | 一种凡纳滨对虾钙离子螯合抗菌肽pv13及其用途 |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
PB01 | Publication | ||
PB01 | Publication | ||
SE01 | Entry into force of request for substantive examination | ||
SE01 | Entry into force of request for substantive examination | ||
GR01 | Patent grant | ||
GR01 | Patent grant |